Nuclear myosin I is a member of the myosin superfamily of actin activated ATPases that is found in the nucleus. There is also abundant evidence for the presence of actin in the nucleus. Nuclear myosin I and actin are necessary for transcription by RNA polymerase I and II. Specifically, actin is necessary for pre-initiation complex formation by RNA polymerase II and actin binds to RNA polymerases I, II and III. We have also found that nuclear myosin I is involved in transcription initiation and that actin and nuclear myosin I are components of elongating complexes. Based on extensive preliminary data, we propose the hypothesis that nuclear myosin I has a crucial role in transcription elongation by RNA polymerase II. Two Specific Aims are proposed to test this hypothesis. Specific Aim 1 has 2 parts. In the first part, we will analyze elongating complexes stalled on an immobilized DNA template to identify binding partners of nuclear myosin I. In the second part, we will restart paused complexes to determine the role of nuclear myosin I in elongation. Specific Aim 2 will directly investigate the possibility that actin and nuclear myosin I have a motor function in transcription elongation. Because RNA polymerase II is also a motor that hydrolyzes ATP, we re-engineered our DNA template so that it contains As only at position 1 and 140. By performing in vitro transcription assays in the presence of CTP and UTP and adding ATP selectively, we will be able to determine the relative contributions of the motor activity of RNA polymerase II and nuclear myosin I to transcription elongation. We will also use a chemical-genetic approach and a nuclear myosin I that is selectively inhibited by N6(2-methylbutyl) ADP to further analyze the motor function of nuclear myosin I in transcription. These experiments are a rigorous and imaginative test of the hypothesis that will provide new and important insights into the molecular basis of transcription. PUBLIC HEALTH RELEVANCE: Transcription, the process of converting DNA into RNA, is one of the most fundamentally important processes in cells. The experiments described in this proposal investigate the role of a new protein in transcription. They have the potential to increase our understanding of the transcription process at the molecular level